The present invention relates to imaging samples and more particularly relates to image samples having a response range that exceeds the measurement range of an imaging system.
Samples that have response ranges that exceed the measurement range of imaging detectors include microarray samples as well as other sample types. A microarray is a tool for analyzing gene expression, such as for matching known and unknown DNA samples, complementary DNA (cDNA) samples, and messenger RNA (mRNA) samples based on base-pairing rules. Nearly every cell of a human body contains a full set of chromosomes and identical genes. At any given time, a fraction of these genes is turned on to perform their genetic purpose. The fraction of genes in a cell that is turned on is typically referred to as being “expressed,” and “gene expression” refers to the subset of genes that is expressed that confers unique properties to each cell type. The term gene expression also refers to the transcription of information contained within DNA into mRNA molecules that are then translated into the proteins that perform the majority of cell functions. The types and amounts of mRNA produced by a cell are studied to identify the particular genes that are expressed, which in turn, provides insight into the ways cells respond to changing environments, changing needs, mutations, and the like. Gene expression is a complex and tightly regulated process that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs. This process acts as both an “on/off” switch to control the genes that are expressed in a cell as well as a “volume control” that increases or decreases the level of expression of particular genes. Microarrays and microarray imaging provide for the detection of genes that are expressed, as well as for the detection of how strongly genes are expressed.
A microarray typically includes a small support structure onto which the sequences of a number of different known genes are-immobilized at fixed locations. These genes are known as probes to which target genes (or targets) might attach. The probes might include DNA, cDNA, or oligonucleotides. An oligonucleotide (or oligo) is a relatively short fragment of a single-stranded DNA that is typically five to fifty nucleotides long. A target may include known and/or unknown DNA, cDNA, mRNA or the like. Support structures often include glass microscope slides, silicon chips, or nylon membranes. The probes may be printed or synthesized directly on a support structure to form the microarray spots of a microarray. Targets that attach with the probes allow researchers to optically identify the targets and the genes that are expressed by a cell and the strength of the expression.
The performance of a microarray experiment is based on hybridization probing. Hybridization probing typically includes targets tagged with fluorescent chromophores to identify complementary probes and targets that are able to base pair with one another. Complementary probes and targets (sometime referred to as mobile probes) are incubated to allow complementary gene sequences to bond together (or hybridize). Bound targets are typically identified using a laser excitation process that causes the fluorescent tags in the targets to fluoresce, emitting known radiation wavelengths that might be in the red and/or green spectral bands. A first excitation-spectral band is often used to excite one set of fluorescent tags coupled to one set of targets and a second excitation-spectral band is often used to excite another set of fluorescent tags coupled to another set of targets. The sets of target may be from a known control sample and a sample having unknown targets. Fluorescent emission (or emission) from the targets provides for the identification of the targets in a sample, as each spot in a microarray includes a known probe that might hybridize with a known complementary target. Moreover, a ratio, for example, of red and green emissions from microarray spots might be used to determine differences in gene expressions, such as gene mutation and the like.
FIG. 1 is a simplified image of a microarray that includes a number of image spots of microarray spots having various emission intensities. As mentioned briefly above, each microarray spot is associated with a particular gene sequence. The image spot locations, relative brightness of the image spots, and/or the colors of the image spots provide an estimate of the gene expression associated with a sample, such as the mRNA of a cell.
Microarray images and images of other samples are typically generated by imaging systems having detectors with fixed measurement range. Emissions from microarray spots often fall within a range of intensities that exceed the fixed measurement range of imaging systems. For example, emission intensities might extent below a threshold detection level and/or above a saturation level of a detector, such as a detector that includes an analog-to-digital (A/D) converter having a fixed measurement range. While imaging systems can be modified to include analog-to-digital converters that have increased measurement ranges, such solutions are often costly. For example, if a detector that includes an AID converter having, say a 12-bit output, is to be changed with an A/D converter having a 32-bit output or a 64-bit output, many components of an imaging system might be updated to accommodate the increased measurement range of the new A/D converter. For example, in addition to changing the A/D converter, a new detector boards on which the new A/D converter is installed might me changed, or an entire computing platform of an imaging system might even be changed to accommodate the increased measurement range of the new A/D converter. Such modifications are costly not only due to the cost of the new components, but are also costly because the imaging system may be unavailable for use during the upgrade period. Moreover, while changing a detector's A/D converter may provide an output having a higher bit width, a higher bit width AID converter may provide a signal wherein the additional bits do not provide increased sample information, but provide bits that represent noise. Accordingly, it is desirable to provide techniques wherein a sample is sufficiently stimulated such that the sample response is sufficiently above the background noise level of the detector to produce a meaningful result and not merely increased noise.
A number of techniques have been used to image samples while avoiding changing detector components (e.g., A/D converters) in imaging system, but tend to be slow and computationally intensive. For example, one traditional technique for collecting a wide range of emission intensities from a sample, and hence collecting a relatively complete set of image data for a sample, using a detector that has a limited analog-to-digital measurement range, includes scanning the sample a sample a number times with different radiation intensities and/or with a radiation detector (e.g., a photomultiplier tube) set to different sensitivity levels for each sample scan. Varying radiation intensity and/or detector sensitivity provides that the measurement range of a detector's A/D converter is not exceeded. However, scanning a sample multiple times typically take a relatively long time. For example, ten scan might be used to collect a relatively full set of image data for a sample. As each scan might take, for example, thirty to fifty minutes, ten scans of the sample will take at least three hundred minutes to five hundred minutes. The time to collect a set of image data might even take longer than this as these times do not take into account the time for changing the radiation intensity of the radiation source and/or adjusting the sensitivity level of a radiation detector.
The foregoing described techniques for collecting a relatively full set of image data for a sample introduce additional difficulties. For example, as sample-scanning times are increased, the increased time of radiation exposure tends photobleach a sample. Therefore, a sample for each scan is not really the same, but has a varying baseline response. Various algorithms might be applied to image data to correct for photobleaching, but such algorithms tend to be complicated and time consuming.
Accordingly, new methods and apparatus are needed for the generation of images of samples using an imaging system having a detector with an A/D converter having a fixed measurement range, wherein sample responses exceed the measurement range of the A/D converter.